Dental materials

Compromised patients Barbara J. Steinberg, DDS, clinical professor of surgery at Drexel University College of Medicine and adjunct associate professor of Oral Medicine at the University of Pennsylvania School of Dental Medicine, will help dentists understand the prevalence, risk factors, prevention and treatment of osteoporosis. Dental materials Gordon J. Christensen, DDS, MSD, PhD, founder and CEO of Practical Clinical Courses, CEO of Clinicians Report Foundation and a practicing prosthodontist in Provo, Utah, will lead a lecture that includes topics such as the state of the profession, CAD/CAM, diagnosis and treatment planning, equipment, esthetic dentistry, laboratory technology, operative dentistry, practice management, fixed prosthodontics and technology.

Some patients with halitosis do not present clinical evidence of oral pathologies or systemic disorders. Stress, depression and anxiety have been identified as risk factors for a shift in oral homeostasis. 4,5 It has been proposed that emotional alterations may be a co-factor in halitosis development. Calil and Marcondes 6 reported increased VSC concentrations in healthy men faced with an anxiety-evoking situation compared with the basal situation, independent of salivary flow.
The relationship between stress and the progression of infectious diseases, including periodontitis, has been the focus of some studies over the last few decades. 4,7,8,9,10 However, the mechanisms involved in this process are still not clear. Potentially pathogenic microorganisms can recognize stress hormones, a fact that has led to the development of the concept of "microbial endocrinology." 11 These microorganisms could use the hormones produced by the host as 'environmental trails' to initiate their growth and pathogenic processes. 12,13,14,15,16,17 In one study, the growth rate of bacteria commonly found in the oral cavity, such as Actinomyces naeslundii, Eikenella corrodens, and Campylobacter gracilis, was stimulated by exposure to noradrenaline. 13 In addition, recent studies show conflicting results of noradrenaline and adrenaline on the growth of P. gingivalis, with studies showing either no effects of these hormones on growth 12,17 or demonstrating a reduction in viability. 13,15 Recently, it was demonstrated that noradrenaline can stimulate the production and activity of arg-gingipain B, an important protease of P. gingivalis. 15 Thus, the effects of stress could be mediated by two potentially synergistic pathways involving the downregulation of the host's defenses and the up-regulation of bacterial pathogenicity.
Because there is clinical evidence that stress is related to halitosis, 6 we hypothesized that periodontal bacteria would respond to stress substances by stimulating their growth and their production of VSC. Because there have been no studies on the effects of stress hormones on VSC production, the aim of the present study was to investigate the effects of catecholamines and cortisol on the growth and VSC production of periodontopathogens.

Methodology substances Used
The following substances were tested: adrenaline -ADR (epinephrine bitartrate, Sigma Chemical Co. ® , St Louis, USA), noradrenaline -NA (arterenol, Sigma ® ) and cortisol -CORT (hydrocortisone, Sigma ® ). The 50 µm concentration of ADR, NA 13,18 and CORT 19 was chosen based on previous studies.The solutions were prepared using distilled water as the solvent and were sterilized using Millex ® (Millipore ® , Billerica, USA) sterile filters with a pore size of 0.22 µm.

Bacterial samples and Culture Conditions
The growth patterns and VSC production of four bacterial species (Porphyromonas endodontalis from clinical specimens -Pe, Porphyromonas gingivalis W83 -Pg, Fusobacterium nucleatum nucleatum ATCC 25586 -Fn and Prevotella intermedia ATCC 25611 -Pi) were examined. These species were selected based on their ability to produce VSC constitutively. The bacteria were grown statically in brain heart infusion broth (BHI; Difco Co., Detroit, USA). To cultivate Pg and Pe, 5 µg/mL hemin (Sigma ® ) and 1 µg/mL menadione (Sigma ® ) were added to the BHI. Bacterial cultures were grown at 37 °C under anaerobic conditions (10% CO 2 , 10% H 2 and 80% N 2 ; MiniMacs Anaerobic Workstation, Don Whitley Scientific ® , Shipley, UK).

study design and experimental Groups
Cultures of Pg, Pe, Pi and Fn were exposed to 50 µM ADR, NOR and CORT for 12 and 24 h (early and mid-logarithmic growth phases). Tubes containing bacteria without the tested substances but containing equivalent volumes of distilled water (vehicle) were used as positive control for growth. Negative controls contained only the tested substances and the culture medium. After incubation, bacterial growth and VSC production were assessed as follows. At least two separate experiments were performed for each hormone tested. In each experiment, 8 replicates were tested for each group (ADR, NA, CORT and controls). 20 susceptibility Tests (Macrodilution Broth Tests) Susceptibility was assayed using a macrodilution broth test as previously described. 21 Tubes containing 5 mL of BHI broth medium (plus 5 µg/mL hemin (Sigma ® ) and 1 µg/mL menadione (Sigma ® ) for the Pg and Pe cultures) were prepared for each experimental group. A standardized bacterial suspension was prepared in sterile saline solution (0.9% NaCl) and adjusted with a spectrophotometer (Unico 1100 RS;Unico ® , Dayton, USA) to an optical density of 0.5 (8 x 10 6 CFU/mL). An inoculum of 500 µL was added to each tube to give a final concentration of 4 x 10 6 CFU/mL. The tubes were then incubated at 37°C in anaerobic conditions for 12 or 24 h. After these periods, bacterial growth was assessed spectrophotometrically at a wavelength of 660 nm.

Bacterial vsC Production Tests
This experiment was conducted as previously described, with some modifications. 20 For bacterial VSC production analyses, the experimental groups and inocula were prepared as described above.
After incubation at 37 °C in anaerobic conditions, 0.1 mL of air from the tubes was aspirated using a 1 mL syringe (BD ® , Franklin Lakes, USA) coupled to a needle (BD ® ) ( Figure 1) and injected into Oral Chroma ® (Abimedical, Osaka, Japan). For a period of 8 minutes, the apparatus performed molecule counts in parts per billion (ppb) of hydrogen sulfite (H 2 S) and methylmercaptan (CH 3 SH). VSC analyses were performed at two different time intervals (12 and 24 h). These incubation lengths were chosen because at these periods, the bacteria are growing exponentially, and there are more viable cells than dead cells.
Gaseous standards of the VSC (H 2 S and CH 3 SH) were prepared as described previously 22

effects of noradrenaline, adrenaline and Cortisol on Bacterial vsC Production
The median values and interquartile deviations of H 2 S and CH 3 SH concentrations (in ppb) for Fn, Pe, Pg and Pi cultures exposed for 12 and 24 h to ADR, NA and CORT are presented in Figures 3 and  4, respectively.
In Fn, only CORT caused a significant increase in H 2 S levels at 12 h (Kruskal-Wallis, p < 0.05), while no changes in H 2 S concentration were observed at 24 h (Kruskal-Wallis, p > 0.05). However, ADR and NA did increase the H 2 S concentration in Fn cultures after 24 h of exposure (p < 0.05). Only Fn showed a reduction in CH 3 SH production by CORT after 24 h (p < 0.05). No other conditions changed the CH 3 SH levels.
H 2 S concentrations were significantly increased only in Pe cultures exposed to NA for 12 h (p < 0.05). No difference in H 2 S production was observed for the groups exposed to ADR and CORT (p > 0.05). In addition, CH 3 SH levels were not altered by exposure of Pe to catecholamines and CORT (p > 0.05).
No statistically significant differences (p > 0.05) were found in H 2 S and CH 3 SH levels among groups and their respective controls in Pg cultured for 12 or 24 h.
Pi was more sensitive to the effects of catecholamines and CORT than the other bacterial cultures. In this case, H 2 S production was up-regulated in the groups exposed to ADR, CORT (12 and 24 h) and NA (24 h) (p < 0.05). None of the experimental groups showed differences in CH 3 SH production compared to their controls (p > 0.05).

discussion
Clinical evidence has shown that stress can increase VSC concentrations, thus contributing to halitosis. 6 The present study provides additional support for the role of stress substances on halitosis, as it was shown that ADR, NOR and CORT can up-regulate the production of VSC by microorganisms commonly found in sub-gingival biofilms.
This study reveals a variation in the growth rates of four periodontopathogenic microorganisms in response to the addition of stress hormones to their culture media. The growth of Fn and Pe was reduced by stress hormones, primarily after 24 h of exposure. In contrast, no significant effects on growth    were observed in Pg and Pi for any of the substances tested. Most of these results are in agreement with the literature regarding the effects of catecholamines on bacterial growth. Roberts et al. 13 demonstrated an inhibitory effect of catecholamines on Fn growth and no effect on the growth of Pi cultures, while Belay et al. 12 and Jentsch et al. 17 showed no change in Pg growth patterns. However, some of the present findings are contrary to previous reports. It was reported that the growth rate of Pg was reduced when exposed to catecholamines. 13,15 Additionally, cortisol was demonstrated to stimulate the growth of Pg cultures. 24 Our results showed no effects of NA, ADR or CORT on the growth rate of this pathogen. There were several differences in the experimental factors used in these studies, including the period of hormone exposure, bacterial strains and culture media used, catecholamine concentration and the method of growth assessment, which may have contributed to the divergent findings.
There are some reports in the literature demonstrating VSC production by periodontopathogenic bacteria. Previous studies revealed that saliva and dental plaque from patients with periodontal disease produces large quantities of VSC, thus contributing to malodor. 25,26,27 Our study shows that periodontal bacteria produce significant VSC levels in vitro and that this production is altered by stress hormones. We observed that the major effects of catecholamines and CORT were on H 2 S levels, mainly in the case of Fn, Pe and Pi. For these bacteria, exposure to these substances caused an increase in H 2 S concentration. Only CORT changed the production of CH 3 SH, and this effect was inhibitory in Fn cultures treated with this glucocorticoid. Given these results, it is possible to assume that the main roles of ADR, NA and CORT are in H 2 S production. To better understand the effects of stress hormones, it will be necessary to utilize enzymatic, genomic and proteomic approaches.
Interestingly, the growth and VSC production of Pg was not affected by any of the stress hormones tested. It is possible that Pg is less sensitive to hormonal influence in the favorable environment of our experiment, which offered a rich-nutrient culture media along with controlled atmosphere and temperature. Previous studies have observed that adrenaline and noradrenaline have strong effects on bacterial growth mainly when the bacteria are cultivated in poor nutrient media. 18,28 For this reason, those authors designed a special medium, serum-SAPI minimal medium, to mimic the challenging in vivo environment. However, this medium was not used in our study. This could be the reason for our observations of slight effects of hormones on growth and VSC production, especially in the case of Pg. It is possible, for example, that these organisms (and others showing negative growth responses) utilize catecholamines for up-regulation of virulence expression rather than for growth 13 and VSC production.
When taking into consideration the times of exposure (12 and 24 h) in our study, differing effects were found. In some experiments, significant effects of the hormones were noted at both times, while the hormone effects were observed at only one time period (12 or 24 h) in other experiments. We expected to observe that a substance that showed an effect at 12 h of exposure would have the same effect at 24 h. As demonstrated, however, this was not always the case. These stress-related substances have short halflives and could be degraded during the time-period of our experiments. Kennedy et al. 29 demonstrated that catecholamines are stable in saliva for up to 2 h at 4 °C. In our study, however, catecholamine concentrations in the culture medium after 12 and 24 h were not determined, and one can imagine that degradation was faster due to the higher temperature. We hypothesized that the bacteria might be able to use this degradation by-product to stimulate VSC production; however, further studies are necessary to elucidate this hypothesis. With regard to the degradation of CORT, it is known that this hormone remains stable in saliva at ambient temperature for up to one week or more 30 and, therefore, would not be degraded during our experimental period.
By comparing the VSC production and growth patterns of bacteria exposed to NA, ADR and CORT, it was possible to observe differing results. These findings suggest a species-specific interaction of ADR, NA and CORT, which leads to differing effects in terms of growth and gas production. It is worth highlighting that, even in some situations where growth was reduced, VSC levels were stimulated. Because of this, it is possible that these stress-related substances have different cellular mechanisms whereby they affect growth and VSC production. Additionally, considering that fewer bacteria could produce higher levels of VSC in the presence of these hormones, it is important to evaluate the clinical importance of this observation. It is possible that these results could be the beginning of an explanation for why patients without advanced periodontal disease or other clinical signs of inflammation have increased complaints of halitosis in stressful situations and have higher VSC levels than non-stressed patients. 6 Investigations of the exposure of catecholamines and cortisol in multi-species biofilm models, animal models for halitosis and clinical studies should be carried out to better understand the relationship of stress and VSC production.

Conclusions
In conclusion, our results indicate that the hormones adrenaline, noradrenaline and cortisol can modulate the growth of periodontopathogenic microorganisms and the production of VSC, thus elucidating another possible mechanism of action for the relationship between stress and the development of halitosis.